Somatotropins are proteins produced by animals which because of their effect on animal growth are commonly referred to as "growth hormones". Historically, growth hormones (somatotropins) were recovered and purified from pituitary gland extracts. With the advent of recombinant DNA technology, somatotropins can now be produced in quantity free of other proteins of natural origins.
Somatotropins produced by recombinant DNA technology in bacteria are commonly accumulated in insoluble refractile bodies in the cytoplasm of the host cell. To recover the somatotropin in its active form requires renaturation which entails solubilization, folding and oxidation of the protein to its native configuration. Examples of renaturation processes are described in detail in European Patent Applications Publication Numbers 114,506A and 192,629A. These processes and subsequent purification processes are usually carried out at high pH. Somatotropins are unstable under these conditions resulting in yield losses, especially when held for prolonged periods of time. Degradation is also temperature dependent with room temperature being worse than 4.degree. C. (the normal temperature used for processing), however, if there were no stability problem, room temperature operation would be better from the processing viewpoint.
It has now been found that the two major decomposition products of somatotropin under these conditions are (1) an isoform of somatotropin with a beta-linked aspartic acid formed from an asparagine located about midway of the protein chain (positions 95-101), and (2) a two chain form of the molecule caused by a break in the polypeptide chain occurring at the asparagine located about midway of the protein chain, i.e. positions 95-101. The break results from the asparagine being converted to a succinimidyl moiety. These forms are often removed during processing resulting in yield loss.